Despite all previous efforts to develop artificial production systems, biological entities such as cells are unrivalled in their ability to produce complex substances such as antibiotics, proteins and nucleic acids. Nearly all substances of cellular origin produced industrially today are extracellular products that are produced within the cell and subsequently excreted into the environment. However, a large proportion of potentially useful substances remain intracellular. In order to release the intracellular material, cells are typically disintegrated by mechanical, physical, chemical or enzymatic means. The recovery efficiency of valuable cellular products is closely linked to their formation, location and their interactions within the biological system. In the past decades, investigations of molecular structures and their functionality within these cellular systems have enhanced the diversification of such products and their large-scale processing. At the same time, requirements regarding product quality, especially in the healthcare sector, resulted in the necessity to deliver well defined, effective and highly pure substances.
Current methods for cell disruption include mechanical and non-mechanical methods. Non-mechanical methods comprise physical (decompression, osmotic shock, thermolysis, freeze drying, microwave), chemical (antibiotics, chelating agents, detergents, solvents, alkalis, supercritical CO2) and enzymatic (lysis, autolysis, phages) methods. On the other hand, mechanical approaches and devices for cell disruption comprise bead milling, homogenisers, cavitation (ultrasonic, hydrodynamic) and microfluidizers. Only some cell disruption methods (mainly mechanical) are performed at an industrial scale, where they are commonly integrated in downstream (e.g. recovery, purification) processing.
For large scale, the most common mechanical methods are bead milling and high pressure homogenisation which are typically implemented as standard operations. When using high pressure homogenisation, a cell solution is forced through a narrow valve under high pressure. By passing through the valve, cells are subjected to turbulence, cavitation, high shear forces and a sudden pressure drop upon discharge which tear the cells apart. The stress and erosion of the valve must be considered in the construction and increase with the rising homogenising pressure. Further, the temperature of the solution rises with increasing pressure, rendering the method energy-consuming and making cooling of the device and the suspension necessary, particularly when temperature-labile enzymes are released. Approximately more than 90% of the power consumed by the homogeniser dissipates as heat, and the cooling cost represents a large portion of the total costs for cell disintegration. Further, successive passages are often required in order to achieve sufficiently high yields (Kula et al., “Purification of Proteins and the Disruption of Microbial Cells.” Biotechnology Progress 1987).
In bead milling, beads are added to a biological fluid which is subsequently subjected to high speed agitation by stirring or shaking. By collision with beads cells are disrupted and intracellular contents are released. Bead milling also produces heat and requires cooling. It is a rather complex process influenced by a variety of parameters, including construction of the bead mill, operational parameters and product specific properties. Construction and geometry of the bead mill are crucial process variables. However, smaller versions are often geometrically dissimilar to industrial-scale bead mills, which complicates the extrapolation of data from laboratory trials to batch performance (Kula et al., “Purification of Proteins and the Disruption of Microbial Cells.” Biotechnology Progress 1987).
Mechanical disruption methods suffer from several drawbacks. Because cells are entirely disrupted, all intracellular materials are released, thereby increasing the contaminants content of the intermediate product. Thus, the product of interest must be separated from a complex mixture of proteins, nucleic acids, and cell fragments. In addition, released nucleic acids may increase the viscosity of the solution and may complicate subsequent processing steps such as chromatography. The cell debris produced by mechanical disintegration often consists of small cell fragments, making the solution difficult to clarify. Complete product release often requires more than one pass through the disruption device, which exacerbates the problem by further reducing the size of the fragments. These are difficult to be removed by continuous centrifugation, because the throughput of the device is inversely related to the square of the particle diameter. Filtration is complicated by the sticky nature of the homogenate and by its tendency to foul membranes. Furthermore, mechanical methods require regular maintenance and costly equipment and are energy consuming. They generate heat and require extensive cooling in order to be usable for temperature-sensitive enzymes. Further, they expose the cells and therefore the extracted products to high shear stress. Most products will be denatured by the heat generated unless the device is sufficiently cooled.
As mentioned above, more selective release methods involve physical, chemical or enzymatic treatment. Chemical treatment involves the use of EDTA, chaotropic agents, organic solvents, antibiotics, acids, alkalis and surfactants. Besides the problem of waste disposal of excess chemicals, these methods are rather expensive and thus not suitable for large-scale application. Contamination of the desired product with the chemicals is another drawback. Some chemicals are not very selective and tend to damage sensitive proteins, enzymes and the cells walls.
Enzymatic cell disruption is more specific but is often limited when applied to complex cell structures with several distinct layers such as bacterial cell walls. Further, it is restricted by the cost of enzyme and buffers. Thus, enzymatic treatment is not applicable at large or industrial scale either. Another drawback is the potential contamination of the desired product with the enzyme.
Physical permeabilization can be accomplished by freeze-thawing or osmotic shock treatment. With freeze-thawing, multiple cycles are necessary for efficient product release, and the process can be quite lengthy. Osmotic shock treatment, on the other hand, may not be sufficient to disrupt cells with robust cell wall structures.
In sum, disadvantages of these methods include comparably high costs, low practicability at large scale, low efficiency and reproducibility, and the necessity to remove added substances after the release.
Taking into account the potential of cellular production systems, there is a need for alternative methods for recovering biomolecules, in particular from cell suspensions, which are easy to handle, cost-efficient, and scalable. Further, the methods should be gentle enough for sensitive biomolecule products and enable a highly selective biomolecule recovery which yields a product with low contamination. It is therefore one objective of the present invention to provide a method or system which overcomes one or more of the above mentioned drawbacks.
It must be noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.
The term “and/or” wherever used herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term”.
The term “about” or “approximately” as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes, however, also the concrete number, e.g., about 20 includes 20.
The term “less than” or “greater than” includes the concrete number. For example, less than 20 means less than or equal to. Similarly, more than or greater than means more than or equal to, or greater than or equal to, respectively.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”.
When used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.
In each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms.
It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.